It was observed that the treatment of 48hr. starved rats with acetate, propionate or butyrate results in large increases in the hepatic Ca2+, Mg2+ and inorganic pyrophosphate (PPi) content apparently due to the formation of calcium and magnesium PPi precipitates within the mitochondrial matrix. The increase in mitochondrial calcium and magnesium was shown to occur using electron probe x-ray microanalysis. Assuming that the free matrix [Mg2+]=1 mM and using the magnesium PPi and calcium PPi solubility products, the free mitochondrial [Ca2+] in the liver was calculated to be 1.2 mM after treatment with short chain fatty acids. This observation was then expanded to all metabolic states and it was concluded that under all in vivo conditions thus far studied that, in the in vivo rat liver, calcium and magnesium PPi precipitates are present in the mitochondrial matrix and the free mitochondrial matrix [Ca2+] is about 1mM. This value is three orders of magnitude greater than values extimated for the free mitochondrial matrix [Ca2+] using isolated mitochondria.